DESCRIPTION: The focus of this research project is to understand how cortactin, an F-actin-binding protein, functions in cell motility and tumor cell invasion. The long-term goal of the proposed research centers on the role of cortactin and associated proteins in lamellipodia function as it pertains to signal transduction and motility, and how cortactin overexpression influences cell motility, adhesion and tumor cell invasiveness. This application specifically addresses the function of cortactin in the organization of the Arp2/3-F-actin based cortical cytoskeleton and how cortactin serves to regulate normal and tumor cell movement. Cortactin is a substrate for multiple tyrosine kinases, including Src, and is over expressed in 30% of head and neck squamous cell carcinomas (HNSCCs). Based on preliminary data, it is posited that cortactin dynamically regulates the cortical actin cytoskeleton by a mechanism that involves tyrosine phosphorylation along with activation and protection of Arp2/3-F-actin networks. The studies described here will use biochemical and cell biological analysis of fibroblast and HNSCC cell lines to explore the hypothesis that cortactin functions to regulate and stabilize cortical Arp2/3-F-actin networks during cell migration, and that enhanced dynamic regulation of Arp2/3-F-actin networks resultant from cortactin overexpression leads to increased invasion of HNSCC. In Aim 1, we will address if cortactin tyrosine phosphorylation affects Arp2/3 actin nucleation activity and its association with Arp2/3-F-actin networks, if cortactin tyrosine phosphorylation influences lamellipodia dynamics, and determine if cortactin protects Arp2/3-F-actin networks from disassembly by ADF/cofilin proteins. Aim 2 will examine cells with reduced or eliminated cortactin expression derived by RNAi to determine how cortactin impacts the cortical actin cytoskeleton, cell migration and adhesion. Aim 3 will examine how cortactin overexpression affects the invasive properties of HNSCC by determining if Arp2/3 complex association and activation are enhanced in HNSCC cell lines overexpressing cortactin, and to determine if cell invasion and extracellular matrix proteolysis is increased as a result of cortactin overexpression in HNSCC. Completion of the proposed Aims will provide a better understanding of how cortactin functions in cell migration and tumor cell invasion/metastasis.